Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five beta-Lactams Simultaneously in Carbapenem-Resistant Klebsiella pneumoniae and Enterobacter cloacae

Abstract

Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While beta-lactam antibiotics are commonly used against Klebsiella pneumoniae and Enterobacter cloacae, there are limited data on OM permeability especially in K. pneumoniae. Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five beta-lactams in carbapenem-resistant K. pneumoniae and E. cloacae. Both clinical isolates harbored a bla(KPC-2) and several other beta-lactamases. The OM permeability of each antibiotic was studied separately (discrete assay) and simultaneously (cassette assay) by determining the degradation of extracellular beta-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses. Our K. pneumoniae isolate was polymyxin resistant, whereas the E. cloacae was polymyxin susceptible. Imipenem penetrated the OM at least 7-fold faster than meropenem for both isolates. Imipenem penetrated E. cloacae at least 258-fold faster and K. pneumoniae 150-fold faster compared to aztreonam, cefepime, and ceftazidime. For our beta-lactams, OM permeability was substantially higher in the E. cloacae compared to the K. pneumoniae isolate (except for aztreonam). This correlated with a higher OmpC porin production in E. cloacae, as determined by proteomics. The cassette and discrete assays showed comparable results, suggesting limited or no competition during influx through OM porins. This cassette assay allowed us, for the first time, to efficiently quantify the OM permeability of multiple beta-lactams in carbapenem-resistant K. pneumoniae and E. cloacae. Characterizing the OM permeability presents a critical contribution to combating the antimicrobial resistance crisis and enables us to rationally optimize the use of beta-lactam antibiotics. IMPORTANCE Antimicrobial resistance is causing a global human health crisis and is affecting all antibiotic classes. While beta-lactams have been commonly used against susceptible isolates of Klebsiella pneumoniae and Enterobacter cloacae, carbapenem-resistant isolates are spreading worldwide and pose substantial clinical challenges. Rapid penetration of beta-lactams leads to high drug concentrations at their periplasmic target sites, allowing beta-lactams to more completely inactivate their target receptors. Despite this, there are limited tangible data on the permeability of beta-lactams through the outer membranes of many Gram-negative pathogens. This study presents a novel, cassette assay, which can simultaneously characterize the permeability of five beta-lactams in multidrug-resistant clinical isolates. We show that carbapenems, and especially imipenem, penetrate the outer membrane of K. pneumoniae and E. cloacae substantially faster than noncarbapenem beta-lactams. The ability to efficiently characterize the outer membrane permeability is critical to optimize the use of beta-lactams and combat carbapenem-resistant isolates.